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DNA sequencing
DNA sequencing by the chemical method of Maxam
and Gilbert (PNAS, 1977)
(formic acid)

• Chemical reagents have been characterized which
  alter one or two bases in DNA.
• An altered base can then be removed from the
  sugar-phosphate backbone of DNA.
• The strand is cleaved with piperidine at the
  sugar residue lacking the base.

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Reading the DNA sequence
Gel PAGE Urea (6 M)
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Sequencing by the chain-terminator or dideoxy
procedure (Sanger, 1977)

• - Enzymatic methods.
• - Random incorporation of a dideoxynucleoside
  triphosphate into a growing strand of DNA. This
  method is an in-vitro DNA synthesis using
  terminators. Incorporation of
  di-deoxynucleotides into growing strand
  terminates synthesis.
• Requires DNA polymerase I. Requires a cloning
  vector with initial primer (M13, high yield
  bacteriophage).

Nobel winner 1980

• - Uses 32P-deoxynucleoside triphosphates.
• Synthesized strand sizes are determined for each
  di-deoxynucleotide by using gel or capillary
  electrophoresis.
• Principle of the method

5
3
T
T
T
T
3
5
primer
ddATP in the reaction anywhere theres a T in
the template strand, occasionally a ddA will be
added to the growing strand
ddA
ddA
ddA
ddA
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The dideoxy chain termination (or enzymatic)
method of DNA sequencing involves the in vitro
synthesis of a DNA strand by a DNA polymerase,
such as ? Klenow fragment of E.coli DNA
polymerase I (used in combination with cloning
the DNA to be sequenced in M13 series of
single-stranded vectors) ? modified form of
phage T7 DNA polymerase, Sequenase. This enzyme,
developed by Tabor and Richardson (P.N.A.S.,
1987, vol. 844767-4772) is a site-directed
mutant (His123?Glu) of bacteriophage gene 5
protein. Features of Sequenase 1. unlike
Klenow fragment, Sequenase can be used with of
double-stranded vectors) 2. reduced
exonuclease activity, 3. highly processive
catalyzing the polymerization of thousands of
nucleotides without dissociating from the
template. ? Taq DNA polymerase (used in cycle
sequencing - PCR)
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Primer walking
La reazione di sequenza permette di stabilire con
buona certezza lordine dei primi 250-350
nucleotidi. Gli inserti di DNA clonati sono
solitamente molto più lunghi (5000
bp). Determinata la sequenza del primo tratto,
si sintetizza un secondo primer disegnato per
ibridarsi con la regione lontana circa 300 basi a
valle del sito di innesco del primo primer. In
maniera simile si sceglie un terzo sito legame
per linnesco, si sintetizza un altro
oligonucleotide e si determina la sequenza delle
successive 250-350 basi. La strategia Primer
walking va avanti fino a completare il
sequenziamento dellintero inserto.
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Chemical method
Enzymatic method
Un apparato per il sequenziamento su gel di
poliacrilamide sottile 0.2-0.4 mm.
MAXAM GILBERT METHOD
SANGER METHOD
? by-pass all the problems associated with
polymerases ? does not require subcloning into
seq. vectors (restriction fragments can be used
directly) ? the only method for sequencing small
oligonucleotides ? time-consuming (labeling of a
single end, purification steps) ? background
due to degradation
? RAPID a large n of samples can be processed
simultaneously ? composition of 2-D structure of
the DNA template can cause premature termination
by DNA polymerase
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Corsa lunga
Corsa breve
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Automated DNA Sequencing

• ? These systems employ fluorescent dyes
  attached to either the primer (I generation of
  this techniques) or the ddNTP (II generation of
  this techniques).
• ? The DNA fragments produced by sequencing
  reactions are run through polyacrylamide gels or
  capillary electrophoresis.
• The detection systems relies on laser-induced
  fluorescence (helium-neon laser 633 nm).

Detecting the bands within the gel is not trivial
as there are only about 10-15 to 10-16 moles
(femtomoles) of DNA in each band.
(laser)
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Proc. Natl. Acad. Sci. USA (1995) vol.92,
pp.4347-4351
Four-dye primer sequencing is one of the most
commonly used method for high-throughput DNA
sequencing. As in other sequencing methodologies,
the detection sensitivity is limited by the
spectroscopic properties of the available dyes
(based on the structure of fluorescein) for
labeling the sequencing fragments.
Structure of FLUORESCEIN and FAM
(5-carboxyfluorescein)
To optimize the absorption and emission
properties of the label, primers have been
developed that exploit fluorescence energy
transfer (ET)
Fluorescence ET (FRET) is mediated by a
dipole-dipole coupling between two chromophores
that results in resonance transfer of excitation
energy from an excited donor molecule to an
acceptor.
Amplified signal
D A
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ET primers have two fluorescent dyes attached.
The effective fluorescence intensity is 2 to 10
times greater than single dye primers. FAM is
selected as common donor, FAM, JOE, TAMRA and ROX
are selected as acceptors.
FAM 5-carboxyfluorescein (SE Succinimidyl ester)
JOE 2,7-dimethoxy-4,5-dichloro- -6-carboxyfluo
rescein R -COOH
ROX 6-carboxy-X-Rhodamine R1 H R2 -COOH
TAMRA tetramethyl-6-carboxyRhodamine R1 H
R2 -COOH
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• A standard procedure
• (II generation sequencing)
• The DNA is prepared as single strand
• A mixture of four normal (deoxy) nucleotides
  (dGTP, dATP, dTTP, dCTP)
• A mixture of four dideoxynucleotides (each
  present in limiting amounts) each labeled with a
  tag that fluoresces a different colour (ddGTP,
  ddATP, ddTTP, ddCTP)
• DNA polymerase
• Adequate buffer

Results can be monitored in real-time on the
interfaced screen and subsequently subjected to
graphically interactive analysis

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READ LENGTHS ? home-made PAGE apparatus (?
17 X 36 cm. - 0.3 mm thick gel) ?
up to 150 - 180 bp ? Macrophor
Electrophoresis Unit (patented design of the
EMBL) LKB-Pharmacia. 20 X 50 cm. -
0.1 mm thick gel. The electrophoresis
unit is equipped with a thermostatic plate that
provides uniform temperature control
(eliminates smiling effects and
resolves G-C compressions) ? up to 300 - 400
bp ? ALF DNA Sequencer Equipped with
fixed-laser detection system, scanning
a polyacrylamide gel (Pharmacia) ? up to
500 bp/hour/lane ? ABI Prism 3700 DNA
Analyzer (Applied Biosystem). Automated
capillary gel electrophoresis system. All four
sequencing reactions are run in a single
capillary (dye-labeled terminator
chemistry). Detect over 500 bases at 98.5
accuracy at 100 bases/hour/capillary. ?
MegaBACE (Amersham-Pharmacia Biotech). DNA
fragments are separated by capillary
electrophoresis (16, 48 or 96-capillary). It is
operated by a confocal scanning laser, and is
capable of up to 12 DNA sequencing runs per
24-hour (read length gt650 bp), producing up to
500.000 bases/day.
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Next Generation Sequencing
Pirosequenziamento
Ronaghi M, Ehleen M and Nyrén P (1998) A
sequencing method based on realtime
pyrophosphate. Science, 238, 363-365.
Si basa sulla rilevazione del pirofosfato
rilasciato dallincorporazione di un nucleotide
durante la sintesi del DNA.

adenosine 5-phosphosulfate (APS)
Apyrase is an ATP diphosphohydrolase. It
catalyses the removal of the gamma phosphate from
ATP and the beta phosphate from ADP. The
phosphate from AMP is not removed. 
PPi is not produced
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Il primer è ibridato allo stampo a singolo
elica, amplificato per PCR, e incubato con gli
enzimi DNA polimerasi, ATP sulfurilasi,
luciferasi e apirasi, adenosin 5 fosfosolfato
(APS) e luciferina. Il primo dei quattro dNTP
viene aggiunto alla reazione. La DNA polimerasi
catalizza lincorporazione del dNTP al filamento
di DNA, se è complementare alla base del
filamento stampo. Ogni evento di incorporazione è
accompagnato dal rilascio di piro-fosfato (PPi)
in quantità equimolare a quella del nucleotide
incorporato. In presenza di adenosina 5
fosfosolfato (APS), lATP sulforilasi converte
quantitativamente il PPi ad ATP, che, a sua volta
guida la conversione, catalizzata dalla
luciferasi, di luciferina ad ossiluciferina con
conseguente produzione di luce di
intensità proporzionale alla quantità di ATP. La
luce prodotta è rilevata da una CCD camera e
visualizzato come picco in un pirogramma.
Lapirasi è un enzima che degrada nucleotidi.
Questo enzima degrada continuamente tutti i dNTP
non incorporati e lATP in eccesso. Lapirasi non
produce PPi. Non appena la degradazione è
completata viene aggiunto un altro dNTP.
I dNTP vengono aggiunti sequenzialmente, uno
alla volta. Poiché il dATP è un substrato
naturale della luciferasi (come la ATP), al suo
posto viene utilizzato la deossiadenosina
a-tio-trifosfato (dATPS) che viene utilizzata
efficentemente dalla DNA polimerasi ma non viene
riconosciuta dalla luciferasi.
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Man mano che il processo continua, il filamento
di DNA complementare è sintetizzato e la sequenza
nucleotidica è determinata dai picchi del
pirogramma ( 300 basi).
La sequenza è TTTGGGGTTGCAGTT ?
DNA polimerasi, apirasi ATP sulforilasi
luciferasi
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454 Technology (Roche)

• To start, the DNA is sheared into 300-800 bp
  fragments, and the ends are polished by
  removing any unpaired bases at the ends.
• Adapters are added to each end. The DNA is made
  single stranded at this point.
• One adapter contains biotin, which binds to a
  streptavidin-coated bead. The ratio of beads to
  DNA molecules is controlled so that most beads
  get only a single DNA attached to them.
• Oil is added to the beads and an emulsion is
  created. PCR is then performed, with each
  aqueous droplet forming its own micro-reactor.
  Each bead ends up coated with about a million
  identical copies of the original DNA.

Biotinylated primers
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• After the emulsion PCR has been performed, the
  oil is removed, and the beads are put into a
  picotiter plate. Each well is just big enough
  to hold a single bead.
• The pyrosequencing enzymes are attached to much
  smaller beads, which are then added to each well.
• The plate is then repeatedly washed with the each
  of the four dNTPs, plus other necessary reagents,
  in a repeating cycle.
• The plate is coupled to a fiber optic chip. A
  CCD camera records the light flashes from each
  well.

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Illumina/Solexa Sequencing

• - This method uses the basic Sanger idea of
  sequencing by synthesis of the second strand of
  a DNA molecule. Starting with a primer, new
  bases are added one at a time, with fluorescent
  tags used to determine which base was added.
• The fluorescent tags block the 3-OH of the new
  nucleotide, and so the next base can only be
  added when the tag is removed.
• So, unlike pyrosequencing, you never have to
  worry about how many adjacent bases of the same
  type are present.
• The cycle is repeated 50-100 times.

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The idea is to put 2 different adapters on each
end of the DNA, then bind it to a slide coated
with the complementary sequences for each primer.
This allows bridge PCR, producing a small spot
of amplified DNA on the slide. The slide contains
millions of individual DNA spots. The spots are
visualized during the sequencing run, using the
fluorescence of the nucleotide being added.
Attached termini
Template DNA PCR Product
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Third generation sequencing
Back in 2003, The Human Genome cost approximately
500 million, years of work and huge
international effort to produce. Actually, the
cost of a genome falls to just 10,000 and maybe
as low as 1000.
Genome Sequencer FLX (Roche analyzer)
Illumina Genome Analyzer
Roche 454 FLX Illumina Genome Analyzer
Amount of starting material needed DNA 3 to 5 µgTotal RNA 20 µg DNA 1 to 5 µgTotal RNA 1 to 2 µg
Sequencing technology Pyrosequencing   Bridge amplification
Read length 200-300 bases  25-35 bases
Sequence yield 100Mb (Mb106) 800Mb-2Gb (Gb109)
Data file 12 to 15Gb/run 1 Tbyte (Tb1012)
Time/run 8hrs 3 to 5 days